Mouse keratinocytes were found to undergo spontaneous "immortalization" without a crisis, in a newly developed serum- free medium, LEP/MK2, consisting of low calcium MEM with non-essential amino acids supplemented with eight factors. Three lines have been isolated to date (MK1, MKDC4, and MK/2057C). The MK1 line has now undergone more than 400 doublings. Giemsa banding has revealed significant karyotypic changes in MK1 as early as the 4th passage, leading to a near- tetraploid karyotype with random loss and gain of individual chromosomes. Minute chromosomes, but no stable markers, have been observed. After these initial changes, the karyotype has remained essentially stable at later passage levels. Line MKDC4 has undergone more than 200 doublings to date and was also found to be subtetraploid at the 7th passage. Line MK/2057C was derived from line MKDC4 at passage 6, found to be resistant to transforming growth factor-beta (TGF-beta) and maintained in LEP/MK2 medium with 1.0 ng/ml TGF-beta. This line has doubled more than 150 times since isolation and remains subtetraploid. Growth parameters were determined for these cell lines at increasing passage levels. Changes with passage level included increased plating efficiency, a reduced requirement of bovine pituitary extract, increased resistance to the growth-inhibitory activity of serum and serum-derived factors including TGF-beta, and decreased response to hormones and growth factors. The establishing lines, like primary and secondary keratinocytes, remain responsive to calcium-induced terminal differentiation and are non-tumorigenic in athymic nude mice. This serum-free system is currently used for transformation studies with oncogenes and chemical carcinogens.